Microtubule assembly and oscillations induced by flash photolysis of caged-GTP

Microtubule assembly and oscillations have been induced using the rapid liberation of GTP by UV flash photolysis of caged-GTP and monitored by time-resolved X-ray scattering. The flash photolysis method of achieving assembly conditions is much faster than the temperature jump method used earlier (msec vs. s range). However, the structural transitions and their rates are similar to those described previously. This means that the rates of the transitions in microtubule assembly observed before are determined by the protein itself, and not by the rate at which assembly conditions are induced. The advantages and limitations of using the photolysis of caged-GTP in microtubule assembly studies are compared with temperature jump methods. Caged-GTP itself reduces the rate of microtubule assembly and oscillations at mM concentrations, consistent with a weak interaction between the nucleotide analogue and the protein. X-rays are capable of slowly liberating GTP and other breakdown products from caged-GTP, even in the absence of UV flash photolysis, thus causing an apparent X-ray-induced microtubule assembly. This effect depends on the X-ray dose but is independent of the caged-GTP concentrations used here (mM range), suggesting that the breakdown of caged-GTP is caused not by the direct absorption of X-rays by the compound but by another intermediate reaction such as the generation of radicals by the X-rays.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-O,O-bis (2-amino ethyl ether)-N,N,N,N-tetraacetic acid - GDP guanosine-5-diphosphate - GTP guanosine-5-triphosphate - caged-GTP P³-1-(2-nitrophenyl) ethyl ester of GTP - HPLC high performance liquid chromatography - Mt-protein microtubule protein (=tubulin +MAPs) - MAP(s) microtubule-associated protein(s) - PC-tubulin phosphocellulose-purified tubulin - PIPES piperazine-1,4-bis(2-ethane sulfonic acid) - UV ultraviolet light Offprint requests to: E. Mandelkow